Hepatitis C virus (HCV) is a recently-identified, enveloped, single- stranded RNA virus that has been shown to be the predominant cause of post-transfusion and sporadically-acquired non-A, non-B hepatitis. Half of all HCV infections progress to chronic hepatitis, and 20% of these lead to cirrhosis. Chronic HCV infection is also associated with the development of hepatocellular carcinoma. Current understanding of the lifecycle of this virus is based largely on inference from its genomic sequence, the deduced structure of the single encoded polyprotein, and knowledge gained from other viral systems. The aims of this project are to extend our understanding of the mechanisms of HCV replication, with particular focus on the activities of viral replicative enzymes, the structural requirements of template RNA, and the role of host cellular factors. Sequence analysis reveals that the HCV polyprotein contains separate structural motifs common to RNA polymerases, RNA helicases, and serine proteases. In preliminary investigations, we have identified and partially purified an RNA-dependent RNA polymerase activity from extracts of infected human hepatocytes. This activity, which is absent from uninfected cell extracts, is capable of replicating HCV RNA sequences by extending a synthetic primer hybridized to the template RNA. We plan to characterize the enzymatic activities of this HCV-specific polymerase, including its products, substrate specificity, cofactor requirements, and susceptibility to competitive and non-competitive inhibitors. In addition, we will use the presence of HCV polymerase activity to identify extrahepatic sites of HCV replication in vivo, to identify cell culture systems as suitable in vitro models of viral replication, and to characterize intracellular RNA replication complexes. These studies should facilitate identification of other virus-encoded and cellular RNA replication complexes. These studies should facilitate identification of other virus-encoded and cellular proteins that support and regulate viral RNA replication, including activities responsible for RNA sequence specificity, initiation of RNA synthesis, and copying fidelity. In separate studies, we will identify, characterize and purify the HCV- specific RNA helicase, an enzyme that likely facilitates RNA replication by catalyzing the unwinding of RNA duplex structures. We will characterize the kinetics, substrate specificity, and cofactor requirements of this enzyme to distinguish it from the variety of helicase gained from these studies will facilitate the design and testing of novel compounds that potently, and selectively, inhibit the replication of this virus. Overall, by extending our understanding of the molecular mechanisms of viral replication, these studies will enhance our ability to interrupt the HCV lifecycle and limit the often severe clinical manifestations of HCV infection.